Avidin is a protein derived from egg white, and streptavidin is a protein derived from Streptomyces avidinii. Avidin and streptavidin each have significantly high affinity (KD=10−16 to 10−14) to biotin (D-[(+)-cis-hexahydro-2-oxo-1H-thieno-(3,4)-imidazole-4-valerate]), and the affinity is one of the most strong interactions between two biological molecules. Their molecular weights are about 60 kDa. Currently, the avidin/streptavidin-biotin interaction is widely applied to the fields of biochemistry, molecular biology, and medicine (Green, (1975), Adv. Protein Chem., 29: 85-133; Green, (1990), Methods Enzymol., 184: 51-67). Avidin and streptavidin each form a tetramer, and one subunit of the tetramer binds to one biotin molecule.
A problem in the use of avidin is non-specific binding thereof. Avidin may non-specifically bind to not only cells but also DNAs, proteins, and biological materials such as membranes. For example, in detection of a material using the avidin-biotin binding, avidin non-specifically binds to materials other than the object material to be detected to increase the background. The reasons for the high non-specific binding of avidin include its high isoelectric point and sugar chains contained in an amount of approximately 10% of the molecular weight. Avidin is a strongly basic protein, having a significantly high isoelectric point of 10 or more, and is positively charged as a whole. Accordingly, it is believed that avidin readily binds to biological materials, which are negatively charged in many cases.
In addition, it is believed that the sugar chains on the surface of avidin easily bind to biological materials (Marttila et al., (2000) FEBS Lett, 467, 31-36). In order to reduce the non-specific binding of avidin, there have been studies on, for example, chemically modified neutravidin in which sugar chains of avidin are removed by glycosidase (Bayer, et al., (1995) Appl Biochem Biotechnol, 53(1), 1-9) and biosynthesis of avidin not receiving sugar chain modification by replacing the asparagine residue at position 17 (a target of glycosilation in avidin), with an isoleucine residue (Marttila, et al., (2000) FEBS Lett, 467, 31-36). In addition, there is a study for reducing the isoelectric point of avidin by converting a lysine residue or an arginine residue of avidin into a neutral amino acid or an acidic amino acid by genetic engineering (Marttila, et al., (1998) FEBS Lett, 441, 313-317).
Although these modifications can reduce non-specific binding of, for example, DNAs and cells to avidin, a reduction in non-specific binding to human sera, which are necessarily used in clinical assay systems, has not been sufficiently investigated. In addition, biosynthesis of avidin mutants needs insect cell expression systems. Accordingly, sequence modification of avidin requires long culture times and high costs and, therefore, has not been put into practical use yet.
According to a reported study relating to affinity between biotin and a biotin-binding protein, such as avidin or streptavidin, binding with fluorescent biotin is strengthened by highly modifying the structure of streptavidin (Aslan, et al., (2005) Proc Natl Acad Sci U.S.A., 102, 8507-8512). Unfortunately, the biotin-binding ability of this protein is severely decreased.
The present inventors purified a protein showing antibacterial activity against Magnaporthe grisea from an edible mushroom, Pleurotus cornucopiae. The protein was revealed to have a biotin-binding activity and was named tamavidin (tamavidin 1). Both the amino acid sequence of the tamavidin 1 protein and the nucleotide sequence of a gene encoding the protein are disclosed in WO 02/072817 (SEQ ID NOs: 1 and 2 in WO02/072817). A homologue (tamavidin 2) of tamavidin 1 was also identified from Plueurotus cornucopiae and was shown to have strong biotin-binding ability. Both the amino acid sequence of the tamavidin 2 protein and the nucleotide sequence of a gene encoding the protein are disclosed in WO 02/072817 (SEQ ID NOs: 3 and 4 in WO02/072817), and a recombinant protein thereof has been successfully produced. Tamavidins 1 and 2 can be expressed in Escherichia coli. In particular, tamavidin 2, which can be easily prepared by purification using an iminobiotin column and has higher heat resistance than that of streptavidin, is an excellent biotin-binding protein. However, although the non-specific binding of tamavidin 2 to nucleic acids and/or proteins is lower than that of existing avidin, it is comparable with that of streptavidin.